Background: Since 2001, we have used conformation sensitive gel electrophoresis (CSGE) as our first choice for F9 gene mutation screening, leading to the identification of about 300 mutations causing haemophilia B (HB). To circumvent the disadvantages of CSGE, we recently evaluated high-resolution melting analysis (HRM), which represents the next-generation mutation scanning technology.
Materials and methods: In order to explore and validate HRM as a new screening method, we analysed 26 HB patients with previous CSGE-detected mutations, 22 patients with CSGE-undetectable mutations and 13 HB patients who had not been previously investigated.
Results: All 61 investigated samples, including the previously investigated and not previously investigated samples, proved to be HRM-positive, with the new screening method showing good efficiency and higher sensitivity than the previously used method. Mixing normal and unknown DNA to generate heterozygote conditions proved an excellent strategy to push the detection performance to its maximum.
Discussion: Mutation scanning by HRM analysis seems to be ideal in our context because it is rapid, cheap and capable of detecting the vast majority of mutations in HB patients. Nevertheless, to improve the detection ability of this scanning technology, it is recommended to start with a good strategy, based on good quality samples and optimised polymerase chain reaction amplification parameters, especially regarding primers and length of the amplicons.